Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer.

PURPOSE: Radiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC). METHODS: Public database was first interrogated to reveal the correlation between CAFs' scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen. RESULTS: CAFs' scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12. CONCLUSION: The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

Druglike, 18F-labeled PET Tracers Targeting Fibroblast Activation Protein.

Fibroblast activation protein (FAP) is a very reliable biomarker for tissue remodeling. FAP has so far mainly been studied in oncology, but there is growing interest in the enzyme in other diseases like fibrosis. Recently, FAP-targeting diagnostics and therapeutics have emerged, of which the so-called FAPIs are among the most promising representatives. FAPIs typically have a relatively high molecular weight and contain very polar, multicharged chelator moieties. While this is not limiting the application of FAPIs in oncology, more druglike FAPIs could be required to optimally study diseases characterized by denser, less permeable tissue. In response, we designed the first druglike 18F-labeled FAPIs. We report target potencies, biodistribution, and pharmacokinetics and demonstrate FAP-dependent uptake in murine tumor xenografts. Finally, this paper puts forward compound 10 as a highly promising, druglike FAPI for 18F-PET imaging. This molecule is fit for additional studies in fibrosis and its preclinical profile warrants clinical investigation.

Head-to-Head Comparison of SSTR Antagonist [68Ga]Ga-DATA5m-LM4 with SSTR Agonist [68Ga]Ga-DOTANOC PET/CT in Patients with Well Differentiated Gastroenteropancreatic Neuroendocrine Tumors: A Prospective Imaging Study.

Neuroendocrine tumors (NETs) are slow-growing tumors that express high levels of somatostatin receptors (SSTRs). Recent studies have shown the superiority of radiolabeled SSTR antagonists in theranostics compared to agonists. In this prospective study, we compared the diagnostic efficacy between [68Ga]Ga-DOTANOC and [68Ga]Ga-DATA5m-LM4 in the detection of primary and metastatic lesions in patients with well differentiated gastroenteropancreatic (GEP) NETs. Histologically proven GEP-NET patients underwent [68Ga]Ga-DOTANOC & [68Ga]Ga-DATA5m-LM4 PET/CT scans, which were analyzed. The qualitative analysis involved the visual judgment of radiotracer uptake validated by the morphological findings using CT, which was considered as the reference standard. Quantitative comparisons were presented as the standardized uptake value (SUV) corrected for lean body mass: SULpeak, SULavg, and tumor-to-background ratios (TBR). In total, 490 lesions were confirmed via diagnostic CT. The lesion-based sensitivity of [68Ga]Ga-DATA5m-LM4 PET/CT was 94.28% (462/490) and 83.46% (409/490) for [68Ga]Ga-DOTANOC PET/CT (p < 0.0001). [68Ga]Ga-DATA5m-LM4 had statistical significance over [68Ga]Ga-DOTANOC in liver metastases [100% vs. 89.4%; p < 0.0001 (292 vs. 253 {283 lesions on CT})] and bone metastases [100% vs. 82.9%; p = 0.005 (45 vs. 34 {41 lesions on CT})]. Statistical significance was also noted for the TBR SULpeak of the primary and liver lesions. [68Ga]Ga-DATA5m-LM4 showed better sensitivity and a higher target-to-background ratio than [68Ga]Ga-DOTANOC PET/CT. [68Ga]Ga-DATA5m-LM4 PET/CT can be used to quantify the extent of skeletal and liver metastases for better planning of SSTR agonist- or antagonist-based therapy.

[177Lu]Lu-DOTAGA.Glu.(FAPi)2 Radionuclide Therapy: a New Treatment Option for Patients with Glioblastoma Multiforme.

In this case report, we present the clinical management of a 52-year-old female patient with a recurrent right temporo-parietal glioblastoma multiforme (GBM). The patient presented with symptoms of headache and loss of balance and recurrence on magnetic resonance imaging (MRI). To evaluate the fibroblast activation protein inhibitor (FAPi) expression in the recurrent lesion, an exploratory [68 Ga]Ga-DOTA.SA.FAPi PET/CT scan was performed. The imaging results revealed FAPi expression in the lesion located in the right temporo-parietal region. Based on the findings of FAPi expression, the patient underwent [177Lu]Lu-DOTAGA.Glu.(FAPi)2 treatment. After completing two cycles of [177Lu]Lu-DOTAGA.Glu.(FAPi)2 therapy, a follow-up [68 Ga]Ga-DOTA.SA.FAPi PET/CT scan was conducted. The post-treatment imaging showed a significant reduction in FAPi uptake and regression in the size of the lesion, as well as a decrease in perilesional edema, as observed on the MRI. Furthermore, the patient experienced an improvement in symptoms and performance status. These results suggest that [68 Ga]Ga-DOTA.SA.FAPi monomer imaging and [177Lu]Lu-DOTAGA.Glu.(FAPi)2 dimer therapeutics hold promise for patients with recurrent GBM when other standard-line therapeutic options have been exhausted. This case highlights the potential of using FAPi-based theranostics in the management of recurrent GBM, providing a potential avenue for personalized treatment in patients who have limited treatment options available.

68Ga-DOTA.SA.FAPi as a Versatile Diagnostic Probe for Various Epithelial Malignancies: A Head-to-Head Comparison with 18F-FDG.

RATIONALE AND OBJECTIVES: Fibroblast Activation Protein (FAP) expressing cancer-associated fibroblasts has been a major breakthrough causing a paradigm shift in targeted theranostics focusing on the tumor microenvironment. In this study, a squaric acid derivative DOTA.SA.FAPi (SA.FAPi) has been evaluated as a potential diagnostic probe in diverse epithelial cancers and compared to the standard-of-care 18F-FDG. METHODS: 25 patients enrolled in this prospective study underwent 18F-FDG and 68Ga-SA.FAPi PET scans on two different days. For biodistribution, standardized uptake values (SUV) were computed by delineating region-of-interest on various body organs. For comparative analysis in disease identification, lesion tracer uptake was quantified using SUVs corrected for lean body mass (SUL), SUVmax, tumor-to-background ratio (TBR) with liver and blood pool as the reference, total lesion glycolysis (TLG for 18F-FDG) and total lesion FAP expression (TLF for 68Ga-SA.FAPi). RESULTS: 25 patients (mean age: 58 ± 8 years) with four types of cancers including hepatocellular carcinoma (HCC, 56% of cohort), gall bladder carcinoma (GB Ca, 12%), adrenocortical carcinoma (ACC, 16%), and breast carcinoma (breast Ca, 16%) were prospectively evaluated. Physiological tracer uptake of 68Ga-SA.FAPi was noted in the salivary glands, thyroid, liver, pancreas, muscles and kidneys with variable uptake in the lacrimal glands, extra-ocular muscles, oral mucosa and uterus. Lesion-based comparative analysis between both the radiotracers demonstrated complete concordant findings in detection of all primary lesions and distant metastases in liver, bones, adrenals and peritoneum whereas discordant findings were noted in lung nodules (20%) and lymph nodes (13%). In overall analysis, 68Ga-SA.FAPi exhibited significantly higher SUVmax (10.3 vs 8.8, p-0.019), SULpeak (6.8 vs 4.9, p-0.000) and SULavg (5.4 vs 4.1, p-0.019) in comparison to 18F-FDG whereas TBR was comparable for both the tracers [TBRLiver: median 1.9 (IQR: 2.6-1.4) vs 1.8 (2.6-1.1), p-0.275; TBRBloodpool: 2.1 (3.7-1.4) vs 2.0 (2.7-1.4), p-0.207]. In subcategorical analysis, 68Ga-SA.FAPi demonstrated higher SUVmax, SULpeak and SULavg values for primary disease (SUVmax: 14.8 (18.7-9.7) vs (12.9-6.6), p-0.087; SULpeak: 8.2 (11.2-6.8) vs 6.3 (8.5-4.4), p-0.037; SULavg: 6.9 ± 2.5 vs 5.1 ± 2.2, p-0.023] and distant metastases (8.8 vs 7.2, p-0.038); 6.3 (8.8-4.4) vs 3.6 (4.4-2.0), p-0.000; 5.4 vs 3.5, p-0.000] whereas comparable values were noted for both the tracers in nodal metastases [9 (13.5-4.1) vs 8 (12.7-4.7), p-0.726; 4.5 (6.2-1.8) vs 4.3 (5.7-2.2), p-0.727; 4.1 ± 2.3 vs 3.7 ± 1.8, p-0.129]. In primary disease, highest 68Ga-SA.FAPi avidity was noted in ACC followed by GB Ca and HCC. In distant metastases, gall bladder, lung and skeletal lesions demonstrated higher 68Ga-SA.FAPi avidity. Moreover, 68Ga-SA.FAPi identified five additional lung lesions which were missed by 18F-FDG in one case of ACC. CONCLUSION: 68Ga-SA.FAPi emerged as an effective, versatile diagnostic probe for imaging various epithelial malignancies similar to 18F-FDG.

Therapeutic potential of [177Lu]Lu-DOTAGA-FAPi dimers in metastatic breast cancer patients with limited treatment options: efficacy and safety assessment.

PURPOSE: The upregulation of fibroblast activation protein (FAP) expression has been observed in various cancers, including metastatic breast carcinoma, prompting research into small molecule inhibitors for both diagnostic and therapeutic purposes. While the diagnostic value of PET/CT imaging using 68 Ga- or 18F-labelled FAPi-monomers in breast cancer diagnosis is well-established, there is a significant need for therapeutic analogs. This retrospective study aimed to assess the safety and effectiveness of [177Lu]Lu-DOTAGA.FAPi dimer radionuclide therapy in patients with advanced-stage breast cancer who had previously undergone [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans to confirm the expression of FAP. MATERIALS AND METHODS: Between November 2020 and March 2023, a compassionate treatment approach was utilized to administer [177Lu]Lu-DOTAGA.FAPi dimer radionuclide therapy to heavily pretreated patients with advanced breast cancer. Nineteen patients (18 females, 1 male) with metastatic breast cancer participated in the study, with an average age of 44.6 ± 10.7 years. The therapy was administered at intervals of 8 to 12 weeks, and the median follow-up duration was 14 months. The primary objective of the study was to assess molecular response using [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans, with response evaluation based on the PERCIST criteria. Secondary endpoints included overall survival (OS), progression-free survival (PFS), clinical response assessment, and safety evaluation using CTCAE v5.0 guidelines. RESULTS: A total of 65 cycles were administered, with a mean cumulative activity of 19 ± 5.7 GBq (510 ± 154 mCi) ranging from 11 to 33.3 GBq (300 to 900 mCi) of [177Lu]Lu-DOTAGA.FAPi dimer. The number of cycles ranged from 2 to 6, with a median of 3 cycles. The treatment protocol consisted of different numbers of cycles administered to the patients: specifically, two cycles were given to five patients, three cycles to nine patients, four cycles to one patient, and six cycles to four patients. Most patients had invasive/infiltrative ductal carcinoma (94.7%), while a small percentage had invasive lobular carcinoma (5.3%). All patients had bone metastases, and five of them also had liver involvement, while seven had brain metastases. Response assessment using [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans showed that 25% of the 16 patients evaluated had partial remission, while 37.5% exhibited disease progression. According to the VAS response criteria, 26.3% achieved complete response, 15.7% had partial response, 42% showed minimal response, 11% had stable disease, and 5% had no response. The clinical disease control rate was promising, with 95% of patients achieving disease control. The clinical objective response rate was 84%. The median follow-up period was 14 months. At the time of analysis, the median overall survival was 12 months, and the median progression-free survival was 8.5 months. Notably, no severe hematological, renal, or hepatic toxicities, electrolyte imbalances, or adverse events of grade 3 or 4 were observed during the study. CONCLUSION: The findings suggest that [177Lu]Lu-DOTAGA.FAPi dimer therapy is well-tolerated, safe, and effective for treating end-stage metastatic breast cancer patients. [177Lu]Lu-DOTAGA.FAPi dimer treatment demonstrated promising efficacy in patients with advanced breast cancer, as indicated by high disease control rates, favorable response outcomes, and acceptable safety profile. Further research and longer follow-up are warranted to assess long-term outcomes and validate these findings.

Head-to-head comparison of [68Ga]Ga-DOTA.SA.FAPi with [18F]F-FDG PET/CT in radioiodine-resistant follicular-cell derived thyroid cancers.

PURPOSE: In the context of radioiodine-resistant follicular-cell derived thyroid cancers (RAI-R-FCTC), [18F]F-FDG PET/CT serves as a widely used and valuable diagnostic imaging method. However, there is growing interest in utilizing molecular imaging probes that target cancer-associated fibroblasts (CAFs) as an alternative approach. This study sought to compare the diagnostic capabilities of [68Ga]Ga-DOTA.SA.FAPi and [18F]F-FDG PET/CT in patients with RAI-R-FCTC. METHODS: In this retrospective study, a total of 117 patients with RAI-R-FCTC were included. The study population consisted of 68 females and 49 males, with a mean age of 53.2 ± 11.7 years. The aim of the study was to perform a comprehensive qualitative and quantitative assessment of [68Ga]Ga-DOTA.SA.FAPi and [18F]F-FDG PET/CT scans in RAI-R-FCTC patients. The qualitative assessment involved comparing patient-based and lesion-based visual interpretations of both scans, while the quantitative assessment included analyzing standardized uptake values corrected for lean body mass (SULpeak and SULavg). The findings obtained from the scans were validated by correlating them with morphological findings from diagnostic computed tomography and/or histopathological examination. RESULTS: Among the 117 RAI-R-FCTC patients, 60 had unilateral local disease, and 9 had bilateral lesions with complete concordance in the detection rate on both PET scans. [68Ga]Ga-DOTA.SA.FAPi had a higher detection rate for lymph nodes (95.4% vs 86.6%, p<0.0001), liver metastases (100% vs. 81.3%, p<0.0001), and brain metastases (100% vs. 39%, p<0.0001) compared to [18F]F-FDG. The detection rates for pleural and bone metastases were similar between the two radiotracers. For lung metastases, [68Ga]Ga-DOTA.SA.FAPi showed a detection rate of 81.7%, whereas [18F]F-FDG had a detection rate of 64.6%. Remarkably, [68Ga]Ga-DOTA.SA.FAPi was able to detect a bowel metastasis that was missed on [18F]F-FDG scan. The median standardized uptake values (SUL) were generally comparable between the two radiotracers, except for brain metastases (SULpeak [68Ga]Ga-DOTA.SA.FAPi vs. [18F]F-FDG: 13.9 vs. 6.7, p-0.0001) and muscle metastases (SULpeak [68Ga]Ga-DOTA.SA.FAPi vs. [18F]F-FDG: 9.56 vs. 5.62, p-0.0085), where [68Ga]Ga-DOTA.SA.FAPi exhibited higher uptake. CONCLUSION: The study results demonstrate the superior performance of [68Ga]Ga-DOTA.SA.FAPi compared to [18F]F-FDG PET/CT in detecting lymph nodal, liver, bowel, and brain metastases in patients with RAI-R-FCTC. These findings highlight the potential of [68Ga]Ga-DOTA.SA.FAPi as a theranostic tool that can complement the benefits of [18F]F-FDG PET/CT in the imaging of RAI-R-FCTC.

Head-to-Head Comparison of [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTANOC Positron Emission Tomography/Computed Tomography Imaging for the Follow-Up Surveillance of Patients with Medullary Thyroid Cancer.

Background: A theranostic probe for accurate staging and treatment is crucial for the management of medullary thyroid cancers (MTCs). The abundance of stroma in most of thyroid cancers, including MTC, opens new avenues for selecting cancer-associated fibroblasts (CAFs) as new molecular imaging and therapeutic targets. [68Ga]Ga-labeled fibroblast activation protein inhibitor (FAPi) molecules have gained importance as alternative molecular imaging agents in the imaging of thyroid cancers. The purpose of this study was to compare the detection efficiency of primary and metastatic lesions of MTCs between [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTANOC positron emission tomography (PET) radiotracers. Materials and Methods: In this retrospective study, [68Ga]Ga-DOTANOC and [68Ga]Ga-DOTA.SA.FAPi PET/CT (computed tomography) images were compared using patient-based and lesion-based analysis in patients with MTC for follow-up assessment. The quantitative assessment included comparing standardized uptake values corrected for lean body mass (SULpeak) and tumor-to-background ratios (TBR). The findings on both scans were validated with the morphological findings of the diagnostic CT. Results: Twenty-seven patients (21 males and 6 females) with a mean age of 42.4 ± 13.2 years (range 14-66 years) were included in the study. [68Ga]Ga-DOTA.SA.FAPi had similar sensitivities as that of [68Ga]Ga-DOTANOC PET/CT for detecting primary tumors (100% [18 of 18] vs. 94.4% [17 of 18], p = 0.979) involved lymph nodes (98.3% [118 of 120] vs. 95% [114 of 120], p = 0.288), and brain metastases (100%). [68Ga]Ga-DOTA.SA.FAPi demonstrated significantly higher sensitivities than [68Ga]Ga-DOTANOC PET/CT for detecting lung nodules (93.5% [87 of 93] vs. 68.9% [64 of 93], p < 0.0001), liver (100% [105 of 105] vs. 46.4% [49 of 105], p < 0.0001), bone (92.4% [110 of 119] vs. 76.5% [91 of 119], p = 0.001), and pleural metastases 98.2% versus 0%. Higher uptake values and TBR values were reported with [68Ga]Ga-DOTA.SA.FAPi compared with that of [68Ga]Ga-DOTANOC. Conclusion: [68Ga]Ga-DOTA.SA.FAPi outperformed [68Ga]Ga-DOTANOC PET/CT in the detection of distant metastases with both patient-based and lesion-based analysis in MTCs.

Inhibition of Poly(ADP-ribose) Polymerase Sensitizes [177Lu]Lu-DOTAGA.(SA.FAPi)2-Mediated Radiotherapy in Triple-Negative Breast Cancer.

Fibroblast activation protein (FAP) is highly expressed in many tumor types and constitutes a promising target for tumor-specific delivery of therapeutic radionuclides. [177Lu]Lu-DOTAGA.(SA.FAPi)2 is a novel radiopharmaceutical based on a novel bidentate inhibitor of FAP that is excreted more slowly than its monomeric counterparts. Still, the efficacy of radiotherapy is mitigated by cascades of DNA damage repair signaling in tumor cells including those via Poly(ADP-ribose) polymerase (PARP). We hereby aimed to evaluate the efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2 in combination with a PARP inhibitor, Olaparib, in the 4T1 murine triple negative breast cancer (TNBC) model. The therapeutic efficacy was visualized using 18F-FDG and [68Ga]Ga-FAPI-04 positron emission imaging/computer tomography (PET/CT). Our results demonstrated that Olaparib suppressed BALB/3T3 fibroblasts in vitro and sensitized the efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2 in mice bearing 4T1 tumors via enhancement of DNA damage. Treatment-associated toxicity was tolerable with only mild leukopenia. Therefore, the combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and Olaparib is a feasible treatment against TNBC.

Head-to-Head Comparison between [68Ga]Ga-DOTA.SA.FAPi and [18F]F-FDG PET/CT Imaging in Patients with Breast Cancer.

This study aimed to compare the diagnostic performance of [68Ga]Ga-DOTA.SA.FAPi with that of [18F]F-FDG PET/CT in detecting primary and metastatic lesions of breast cancer. [18F]F-FDG and [68Ga]Ga-DOTA.SA.FAPi PET/CT scans of histologically proven breast cancer patients were compared according to patient-based and lesion-based analysis. Forty-seven patients with a mean age of 44.8 ± 9.9 years (range: 31-66 years) were evaluated. A total of 85% of patients had invasive ductal carcinoma, and 15% had invasive lobular carcinoma. The tracer uptake [SULpeak, SULavg, and the median tumor-to-background ratio (TBR)] was significantly higher in [68Ga]Ga-DOTA.SA.FAPi than with [18F]F-FDG PET/CT for lymph nodes, pleural metastases, and liver lesions (p < 0.05). However, for brain metastasis, only the median TBR was significantly higher (p < 0.05) compared to [18F]F-FDG. In patient-based analysis the sensitivity of [68Ga]Ga-DOTA.SA.FAPi PET/CT was higher, but not significant than that of [18F]F-FDG PET/CT in the detection of both primary tumors and metastatic lesions. According to lesion-based analysis, on diagnostic CT, 47 patients had 44 primary tumors, 248 lymph nodes, 15 pleural, 88 liver, and 42 brain metastases. [68Ga]Ga-DOTA.SA.FAPi scan identified more abnormal lesions than [18F]F-FDG in all the primary and metastatic sites with a maximum marked difference in the primary site [88.6% vs. 81.8%; p-0.001], lymph nodes [89.1% vs. 83.8%; p-0.0001], pleural metastases [93.3% vs. 73%; p-0.096] and brain metastasis [100% vs. 59.5%; p-0.0001]. [68Ga]Ga-DOTA.SA.FAPi PET/CT was superior to [18F]F-FDG PET/CT in the imaging of breast cancers.

[111In]In/[177Lu]Lu-AAZTA5-LM4 SST2R-Antagonists in Cancer Theranostics: From Preclinical Testing to First Patient Results.

Aiming to expand the application of the SST2R-antagonist LM4 (DPhe-c[DCys-4Pal-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH2) beyond [68Ga]Ga-DATA5m-LM4 PET/CT (DATA5m, (6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate), we now introduce AAZTA5-LM4 (AAZTA5, 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-[pentanoic-acid]perhydro-1,4-diazepine), allowing for the convenient coordination of trivalent radiometals of clinical interest, such as In-111 (for SPECT/CT) or Lu-177 (for radionuclide therapy). After labeling, the preclinical profiles of [111In]In-AAZTA5-LM4 and [177Lu]Lu-AAZTA5-LM4 were compared in HEK293-SST2R cells and double HEK293-SST2R/wtHEK293 tumor-bearing mice using [111In]In-DOTA-LM3 and [177Lu]Lu-DOTA-LM3 as references. The biodistribution of [177Lu]Lu-AAZTA5-LM4 was additionally studied for the first time in a NET patient. Both [111In]In-AAZTA5-LM4 and [177Lu]Lu-AAZTA5-LM4 displayed high and selective targeting of the HEK293-SST2R tumors in mice and fast background clearance via the kidneys and the urinary system. This pattern was reproduced for [177Lu]Lu-AAZTA5-LM4 in the patient according to SPECT/CT results in a monitoring time span of 4-72 h pi. In view of the above, we may conclude that [177Lu]Lu-AAZTA5-LM4 shows promise as a therapeutic radiopharmaceutical candidate for SST2R-expressing human NETs, based on previous [68Ga]Ga-DATA5m-LM4 PET/CT, but further studies are needed to fully assess its clinical value. Furthermore, [111In]In-AAZTA5-LM4 SPECT/CT may represent a legitimate alternative diagnostic option in cases where PET/CT is not available.

Novel Generation of FAP Inhibitor-Based Homodimers for Improved Application in Radiotheranostics.

Radiopharmaceuticals based on the highly potent FAP inhibitor (FAPi) UAMC-1110 have shown great potential in molecular imaging, but the short tumor retention time of the monomers do not match the physical half-lives of the important therapeutic radionuclides 177Lu and 225Ac. This was improved with the dimer DOTAGA.(SA.FAPi)2, but pharmacological and radiolabeling properties still need optimization. Therefore, the novel FAPi homodimers DO3A.Glu.(FAPi)2 and DOTAGA.Glu.(FAPi)2. were synthesized and quantitatively radiolabeled with 68Ga, 90Y, 177Lu and 225Ac. The radiolabeled complexes showed high hydrophilicity and were generally stable in human serum (HS) and phosphate-buffered saline (PBS) at 37 °C over two half-lives, except for [225Ac]Ac-DOTAGA.Glu.(FAPi)2 in PBS. In vitro affinity studies resulted in subnanomolar IC50 values for FAP and high selectivity for FAP over the related proteases PREP and DPP4 for both compounds as well as for [natLu]Lu-DOTAGA.Glu.(FAPi)2. In a first proof-of-principle patient study (medullary thyroid cancer), [177Lu]Lu-DOTAGA.Glu.(FAPi)2 was compared to [177Lu]Lu-DOTAGA.(SA.FAPi)2. High uptake and long tumor retention was observed in both cases, but [177Lu]Lu-DOTAGA.Glu.(FAPi)2 significantly reduces uptake in non-target and critical organs (liver, colon). Overall, the novel FAPi homodimer DOTAGA.Glu.(FAPi)2 showed improved radiolabeling in vitro and pharmacological properties in vivo compared to DOTAGA.(SA.FAPi)2. [177Lu]Lu-DOTAGA.Glu.(FAPi)2 and [225Ac]Ac-DOTAGA.Glu.(FAPi)2 appear promising for translational application in patients.

68 Ga-DOTA.SA.FAPI as a Potential, Noninvasive Diagnostic Probe for Recurrent and Metastatic Adrenocortical Carcinoma : A Head-to-Head Comparison With 18F-FDG.

ABSTRACT: Metastatic or recurrent adrenocortical carcinoma (ACC) is a potentially fatal malignancy, which poses major challenges in disease management owing to lack of effective systemic therapies. The drastically reduced survival rates require prompt identification of selective molecules for development of targeted therapeutics. We evaluated the squaric acid containing FAPI derivative, DOTA.SA.FAPI (FAPI), as a potential diagnostic probe in 2 cases of histopathologically proven metastatic and recurrent ACC. Both patients underwent 18 F-FDG and 68 Ga-FAPI PET/CT scans for comparative analysis. 68 Ga-DOTA.SA.FAPI emerged as an excellent diagnostic agent for ACC and performed similar to 18 F-FDG.

[68Ga]Ga-DATA5m-LM4, a PET Radiotracer in the Diagnosis of SST2R-Positive Tumors: Preclinical and First Clinical Results.

Radiolabeled somatostatin subtype 2 receptor (SST2R)-antagonists have shown advantageous profiles for cancer theranostics compared with agonists. On the other hand, the newly introduced hybrid chelator (6-pentanoic acid)-6-(amino)methyl-1,4-diazepinetriacetate (DATA5m) rapidly binds Ga-68 (t1/2: 67.7 min) at much lower temperature, thus allowing for quick access to "ready-for-injection" [68Ga]Ga-tracers in hospitals. We herein introduce [68Ga]Ga-DATA5m-LM4 for PET/CT imaging of SST2R-positive human tumors. LM4 was obtained by 4Pal3/Tyr3-substitution in the known SST2R antagonist LM3 (H-DPhe-c[DCys-Tyr-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH2) and DATA5m was coupled at the N-terminus for labeling with radiogallium (Ga-67/68). [67Ga]Ga-DATA5m-LM4 was evaluated in HEK293-SST2R cells and mice models in a head-to-head comparison with [67Ga]Ga-DOTA-LM3. Clinical grade [68Ga]Ga-DATA5m-LM4 was prepared and injected in a neuroendocrine tumor (NET) patient for PET/CT imaging. DATA5m-LM4 displayed high SST2R binding affinity. [67Ga]Ga-DATA5m-LM4 showed markedly higher uptake in HEK293-SST2R cells versus [67Ga]Ga-DOTA-LM3 and was stable in vivo. In HEK293-SST2R xenograft-bearing mice, it achieved longer tumor retention and less kidney uptake than [67Ga]Ga-DOTA-LM3. [68Ga]Ga-DATA5m-LM4 accurately visualized tumor lesions with high contrast on PET/CT. In short, [68Ga]Ga-DATA5m-LM4 has shown excellent prospects for the PET/CT diagnosis of SST2R-positive tumors, further highlighting the benefits of Ga-68 labeling in a hospital environment via the DATA5m-chelator route.

Novel Fibroblast Activation Protein Inhibitor-Based Targeted Theranostics for Radioiodine-Refractory Differentiated Thyroid Cancer Patients: A Pilot Study.

Background: This exploratory study was meant to assess clinical and safety data with a novel fibroblast activation protein inhibitor-based targeted theranostics as a salvage treatment option in radioiodine-refractory differentiated thyroid cancer (RR-DTC) patients who had progressed on tyrosine kinase inhibitors. Methods: Patients with metastatic RR-DTC who progressed on sorafenib/lenvatinib were prospectively recruited. If [68Ga]Ga-DOTA.SA.FAPi positron emission tomography/computed tomography scan demonstrated moderate-to-excellent uptake in metastases, and patients had given informed consent, they received intravenous [177Lu]Lu-DOTAGA.(SA.FAPi)2 as therapy at eight-weekly intervals. The primary endpoints were thyroglobulin (Tg) response and functional imaging response. The secondary endpoints were visual analog score (VAS) and Eastern Cooperative Oncology Group (ECOG) performance status. The grading of toxicities was performed by using Common Terminology Criteria for Adverse Events (CTCAEV5.0). The sequential images were acquired by a dual-headed gamma camera, and dosimetric calculations were performed by using OLINDA/EXM V2.1. Results: Fifteen patients were recruited [age: 55 ± 9 years (range: 39-67)]. [177Lu]Lu-DOTAGA.(SA.FAPi)2 had median whole-body Teff of 88.06 hours (interquartile range [IQR]: 86.6-99). The colon was identified as a critical organ. The whole-body effective dose was 1.62E-01 ± 1.53E-02 mSv/MBq. A total of 45 cycles were administered, and the median cumulative administered activity was 8.2 ± 2.7 GBq (range 5.5-14 GBq). The median absorbed doses to the tumor lesions were 1.08E+01 (IQR: 4.16E+00 to 8.97E+01) mSv/MBq per cycle. The Serum Tg level significantly decreased after treatment [(median Tg: baseline-10,549 ng/mL (IQR: 3066.5-39,450) versus at the time of assessment: 5649 ng/mL (IQR: 939.5-17,099), p = 0.0005)]. Molecular response assessment revealed no complete response; however, partial response was documented in four, and stable disease in three patients. The VASmax scores [pre-therapy: 9 (IQR: 8-10) versus follow-up: 6 (3-6) (p-0.0001)], and ECOG [3, (IQR: 2-3 vs. 2, (IQR: 2-3) (p-0.0078)] performance scores significantly improved after treatment. None of the patients experienced grade III/IV hematological, renal, or hepatotoxicity. Conclusion: These preliminary data suggest that the novel molecule [177Lu]Lu-DOTAGA.(SA.FAPi)2 is safe, seems effective, and, most importantly, opens up a new avenue for the treatment of aggressive RR-DTC patients who have exhausted all standard line of treatments.

First-In-Human Results on the Biodistribution, Pharmacokinetics, and Dosimetry of [177Lu]Lu-DOTA.SA.FAPi and [177Lu]Lu-DOTAGA.(SA.FAPi)2.

Recently, great interest has been gained regarding fibroblast activation protein (FAP) as an excellent target for theranostics. Several FAP inhibitor molecules such as [68Ga]Ga-labelled FAPI-02, 04, 46, and DOTA.SA.FAPi have been introduced and are highly promising molecular targets from the imaging point of view. FAP inhibitors introduced via bifunctional DOTA and DOTAGA chelators offer the possibility to complex Lutetium-177 due to an additional coordination site, and are suitable for theranostic applications owing to the increased tumor accumulation and prolonged tumor retention time. However, for therapeutic applications, very little has been accomplished, mainly due to residence times of the compounds. In an attempt to develop a promising therapeutic radiopharmaceutical, the present study aimed to evaluate and compare the biodistribution, pharmacokinetics, and dosimetry of [177Lu]Lu-DOTA.SA.FAPi, and [177Lu]Lu-DOTAGA.(SA.FAPi)2 in patients with various cancers. The FAPi agents, [177Lu]Lu-DOTA.SA.FAPi and [177Lu]Lu-DOTAGA.(SA.FAPi)2, were administered in two different groups of patients. Three patients (mean age-50 years) were treated with a median cumulative activity of 2.96 GBq (IQR: 2.2-3 GBq) [177Lu]Lu-DOTA.SA.FAPi and seven (mean age-51 years) were treated with 1.48 GBq (IQR: 0.6-1.5) of [177Lu]Lu-DOTAGA.(SA.FAPi)2. Patients in both the groups underwent serial imaging whole-body planar and SPECT/CT scans that were acquired between 1 h and 168 h post-injection (p.i.). The residence time and absorbed dose estimate in the source organs and tumor were calculated using OLINDA/EXM 2.2 software. Time versus activity graphs were plotted to determine the effective half-life (Te) in the whole body and lesions for both the radiotracers. Physiological uptake of [177Lu]Lu-DOTA.SA.FAPi was observed in the kidneys, colon, pancreas, liver, gall bladder, oral mucosa, lacrimal glands, and urinary bladder contents. Physiological biodistribution of [177Lu]Lu-DOTAGA.(SA.FAPi)2 involved liver, gall bladder, colon, pancreas, kidneys, and urinary bladder contents, lacrimal glands, oral mucosa, and salivary glands. In the [177Lu]Lu-DOTA.SA.FAPi group, the highest absorbed doses were noted in the kidneys (0.618 ± 0.015 Gy/GBq), followed by the colon (right colon: 0.472 Gy/GBq and left colon: 0.430 Gy/GBq). In the [177Lu]Lu-DOTAGA.(SA.FAPi)2 group, the colon received the highest absorbed dose (right colon: 1.160 Gy/GBq and left colon: 2.870 Gy/GBq), and demonstrated a significantly higher mean absorbed dose than [177Lu]Lu-DOTA.SA.FAPi (p < 0.011). [177Lu]Lu-DOTAGA.(SA.FAPi)2 had significantly longer median whole-body Te compared to that of [177Lu]Lu-DOTA.SA.FAPi [46.2 h (IQR: 38.5-70.1) vs. 23.1 h (IQR: 17.8-31.5); p-0.0167]. The Te of tumor lesions was significantly higher for [177Lu]Lu-DOTAGA.(SA.FAPi)2 compared to [177Lu]Lu-DOTA.SA.FAPi [86.6 h (IQR: 34.3-94.6) vs. 14 h (IQR: 12.8-15.5); p-0.0004]. The median absorbed doses to the lesions were 0.603 (IQR: 0.230-1.810) Gy/GBq and 6.70 (IQR: 3.40-49) Gy/GBq dose per cycle in the [177Lu]Lu-DOTA.SA.FAPi, and [177Lu]Lu-DOTAGA.(SA.FAPi)2 groups, respectively. The first clinical dosimetry study demonstrated significantly higher tumor absorbed doses with [177Lu]Lu-DOTAGA.(SA.FAPi)2 compared to [177Lu]Lu-DOTA.SA.FAPi. [177Lu]Lu-DOTAGA.(SA.FAPi)2 is safe and unveiled new frontiers to treat various end-stage cancer patients with a theranostic approach.

Fibroblast activation protein inhibitor (FAPi) positive tumour fraction on PET/CT correlates with Ki-67 in liver metastases of neuroendocrine tumours.

AIM: Gallium-68-labelled inhibitors of the fibroblast activation protein (FAPi) enable positron emission tomography/computed tomography (PET/CT) imaging of fibroblast activation. We evaluated if [68Ga]Ga-DATA5m.SA.FAPi PET/CT is related to Ki-67 as a marker of tumour aggressiveness in patients with liver metastases of NET. METHODS: Thirteen patients with liver metastases of a histologically confirmed NET who underwent PET/CT with [68Ga]Ga-DATA5m.SA.FAPi, [18F]FDG and [68Ga]Ga-DOTA-TOC were retrospectively analyzed. PET-positive liver tumour volumes were segmented for calculation of volume, SUVmax and PET-positive tumour fraction (TF). PET parameters were correlated with Ki-67. RESULTS: FDGSUVmax correlated positively (rho = 0.543, p < 0.05) and DOTATOCSUVmax correlated negatively (rho = -0.618, p < 0.05) with Ki-67, the correlation coefficients were in the moderate range. There was no significant correlation between FAPiSUVmax and Ki-67 (rho = 0.382, p > 0.05). FAPiTF correlated positively (rho = 0.770, p < 0.01) and DOTATOCTF correlated negatively (rho = -0.828, p < 0.01) with Ki-67, both significantly with high correlation coefficients. FDGTF also correlated significantly with Ki-67, with a moderate correlation coefficient (rho = 0.524, p < 0.05). The ratio FAPiVOL:DOTATOCVOL showed a significant and strong correlation with Ki-67 (rho = 0.808, p < 0.01). CONCLUSION: The ratio FAPiVOL:DOTATOCVOL might serve as a clinical parameter for the assessment of dedifferentiation and aggressiveness of liver metastases in patients with NET. [68Ga]Ga-DATA5m.SA.FAPi might hold potential for identification of high-risk patients. Further studies are warranted to evaluate its prognostic significance in comparison to [18F]FDG in patients with NET.

Squaric Acid-Based Radiopharmaceuticals for Tumor Imaging and Therapy.

Targeting vectors bound to a chelator represent a significant fraction of radiopharmaceuticals used nowadays for diagnostic and therapeutic purposes in nuclear medicine. The use of squaramides as coupling units for chelator and targeting vector helps to circumvent the disadvantages of several common coupling methods. This review gives an overview of the use of squaric acid diesters (SADE) as linking agents. It focuses on the conjugation of cyclic chelators, e.g., DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), as well as hybrid chelators like AAZTA5 (6-pentanoic acid-6-amino-1,4-diazepine tetracetic acid) or DATA5m (6-pentanoic acid-6-amino-1,4-diazapine-triacetate) to different targeting vectors, e.g., prostate-specific membrane antigen inhibitors (KuE; PSMAi), fibroblast activation protein inhibitors (FAPi), and monoclonal antibodies (mAbs). An overview of the synthesis, radiolabeling, and in vitro and in vivo behavior of the described structures is given. The unique properties of SADE enable a fast and simple conjugation of chelators to biomolecules, peptides, and small molecules under mild conditions. Furthermore, SA-containing conjugates could not only display similar in vitro characteristics in terms of binding affinity when compared to reference compounds, but may even induce beneficial effects on the pharmacokinetic properties of these radiopharmaceuticals.

Fibroblast Activation Protein (FAP) targeting homodimeric FAP inhibitor radiotheranostics: a step to improve tumor uptake and retention time.

Several radiopharmaceuticals targeting fibroblast activation protein (FAP) based on the highly potent FAP inhibitor UAMC1110 are currently under investigation. Pre-clinical as well as clinical research exhibited the potential of these imaging agents. However, the monomeric small molecules seemed to have a short retention time in the tumor in combination with fast renal clearance. Therefore, our strategy was to develop homodimeric systems having two FAP inhibitors to improve residence time and tumor accumulation. The homodimers with two squaramide coupled FAP inhibitor conjugates DOTA.(SA.FAPi)2 and DOTAGA.(SA.FAPi)2 were synthesized and radiochemically evaluated with gallium-68. [68Ga]Ga-DOTAGA.(SA.FAPi)2 was tested for its in vitro stability, lipophilicity and affinity properties. In addition, human PET/CT scans were performed for [68Ga]Ga-DOTAGA.(SA.FAPi)2 with a head-to-head comparison with [68Ga]Ga-DOTA.SA.FAPi and [18F]FDG. Labeling with gallium-68 demonstrated high radiochemical yields. Inhibition measurements revealed excellent affinity and selectivity with low nanomolar IC50 values for FAP. In PET/CT human studies, significantly higher tumor uptake as well as longer tumor retention could be observed for [68Ga]Ga-DOTAGA.(SA.FAPi)2 compared to [68Ga]Ga-DOTA.SA.FAPi. Therefore, the introduction of the dimer led to an advance in human PET imaging indicated by increased tumor accumulation and prolonged retention times in vivo and thus, the use of dimeric structures could be the next step towards prolonged uptake of FAP inhibitors resulting in radiotherapeutic analogs of FAP inhibitors.